key: cord-0742722-65mmlmks
authors: Campos, Gubio S.; Sardi, Silvia I.; Falcao, Melissa B.; Belitardo, Emilia M.M.A.; Rocha, Danilo J.P.G.; Rolo, Carolina A.; Menezes, Aline D.; Pinheiro, Carina S.; Carvalho, Rejane H.; Almeida, João P.P.; Aguiar, Eric R.G.R.; Pacheco, Luis G.C.
title: Ion torrent-based nasopharyngeal swab metatranscriptomics in COVID-19
date: 2020-05-21
journal: J Virol Methods
DOI: 10.1016/j.jviromet.2020.113888
sha: cb9a1a77b12ae0f62ba193961e49866637772cd7
doc_id: 742722
cord_uid: 65mmlmks

Herein, we describe the detection of a SARS-CoV-2 genome through metatranscriptome next-generation sequencing directly from the nasopharyngeal swab of a suspected case of local transmission of Covid-19, in Brazil. Depletion of human ribosomal RNA and use of an optimized in-house developed bioinformatics strategy contributed to successful detection of the virus.

using real-time reverse transcription PCR (RT-qPCR) for diagnosing COVID-19 (3, 4) . Possible interfering factors in RT-qPCR may arise from high viral recombination of coronaviruses (5) and varying viral loads in the natural course of disease (6) , but this still needs further studies in the COVID-19 diagnostics context (7) . Therefore, developing new protocols for untargeted clinical metagenomics may provide valuable diagnostic tools for detecting SARS-CoV-2 and other pathogens in a current global scenario of emerging viral infections. Other studies have recently reported on metagenome-based strategies for next-generation sequencing of SARS-CoV-2 and varying results were reached, when comparing amplicon-based sequencing or direct metatranscriptome sequencing. Also, metagenome sequencing from bronchoalveolar lavage specimens rendered better results when compared to nasal swabs (5, 8) .

In this study, we successfully detected a SARS-CoV-2 genome directly from a nasopharyngeal swab specimen of a suspected case of local transmission of COVID-19 in Brazil, using a metatranscriptomics approach. On March 03, 2020, a female patient presented to a public hospital, in Feira de Santana-Bahia, Brazil, complaining of sore-throat and mild-dyspnea. Due to self-reported close contact with a returning traveler from Italy who had a confirmed COVID-19 diagnosis, specific diagnostic investigation was warranted, and a nasopharyngeal swab sample was immediately collected. Viral RNA extraction and RT-qPCR amplification were carried out following a protocol validated in our group at the Institute of Health Sciences, Federal University of Bahia, Salvador-BA, Brazil, according to a Promega application report (Promega Application Report, 2020; https://www.promega.com.br/applications/virus-detection-assay-coronavirusdetection-covid-19-sars-cov-2/). In brief, 200 µL of samples stored in transport medium In parallel, the extracted RNA (ca. 14 ng/µL) was submitted to metatranscriptome nextgeneration sequencing in the Ion S5 platform (ThermoFisher) using an Ion 540™ chip and the Ion Total RNA-Seq kit v2, as per the manufacturer's protocol (ThermoFisher). One sample was previously processed with the Low Input RiboMinus™ Eukaryote System v2 (ThermoFisher), for depletion of human ribosomal RNA from total RNA (Fig. 1) . Sequence reads were pre-processed by quality filtering (Phred >20), presence of ambiguous bases (filtering out of reads with >1% Ns), and size (reads >= 30 nt); all reads deriving from human transcripts were also discarded: 77.29%

in the rRNA depleted library and 84.49% in the whole RNA library. The remaining sequence reads (size range: 30 -358 nt) were then used for contig assembly (see detailed pipeline in Fig. 1 ).

Sequencing data is available at NCBI's SRA database under accession number PRJNA613951.

Overall, depletion of human rRNA rendered significantly higher numbers of contigs (n = 51; N50 = 180 nt) confidentially aligned to the SARS-CoV-2 viral genome (Figure 1b) , yielding a genome coverage of 29.9% for contigs (as opposed to 5.4 % for the non-depleted sample) and 59.9% for total reads, which is higher than the coverage reported for nasopharyngeal swabs in recent studies (8) . SNP analysis using Atlas 2 (https://sourceforge.net/p/atlas2/wiki/Atlas-SNP/) where reads larger than 30nt with Phred quality > 20 were aligned into human reference genome.

Unaligned reads were used to perform contig assemblage. Assembled contigs were compared to NCBI databases using Blast software. Contigs that presented sequence similarity to SARS-Cov2-2 with e-value lower than 1e-5 were considered as from viral origin. Viral contigs were further submitted to contig extension using SPAdes and 'trusted contigs' option that was followed by cap3 tool to remove sequence redundancy. B) SARS-Cov-2 coverage profile of reads and assembled contigs. Reads were normalized by number of reads from each library. Contigs in red indicate high quality contigs larger than 400nt with coverage of reads in both libraries. 

particularly regarding depletion of ribosomal RNA, may significantly impact metatranscriptomic results. CRediT author statement GSC: Investigation, Resources, Project administration, Supervision, Writing -Review & Editing

Writing -Review & Editing; MBF: Investigation, Supervision; EMMAB: Investigation, Formal analysis; DJPGR: Investigation, Formal analysis; CAR: Investigation, Formal analysis; ADM: Investigation

Formal analysis; JPPA: Investigation, Methodology, Software; ERGRA: Conceptualization, Methodology, Writing -Original Draft, Writing -Review & Editing, Funding, Supervision; LGCP: Conceptualization, Methodology, Writing -Original Draft

Routes for COVID-19 importation in Brazil

MedRxiv Infectious Diseases (except HIV/AIDS) Mar

2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel

This study was partially funded by the following research grants: 

The authors declare that there is no conflict of interest related to this article content.